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A liquid chromatographic (LC) method was validated for the determination of total vitamin B6 in infant formula. Total vitamin B6 was quantified by converting the phosphorylated and free vitamers into pyridoxine. Pyridoxine was determined by ion pair reversed-phase LC with fluorescence detection. The method was subjected to an AOAC collaborative study involving a factory-manufactured, milk- and soy-based infant formula. Each was spiked at 3 concentrations in the range of 0–1 μg/g and sent as blind duplicate to participant laboratories. Nine laboratories returned valid data which were statistically analyzed for outliers and precision parameters. The repeatability relative standard deviation (RSDr) ranges were 2.0–4.0 and 3.5–5.9% for fortified milk- and soy-based formulas, respectively. The reproducibility relative standard deviation (RSDR) ranges were 8.2–8.4 and 6.7–11.2% for fortified milk- and soy-based formulas, respectively. HORRAT values ranged from 0.42 to 0.53, indicating that the precision of the method is acceptable. The mean RSDr:RSDR values were 0.60 and 0.55 for milk- and soy-based formulas, respectively. As expected, RSDs for the unfortified samples were higher, but their HORRAT values (0.81 and 2.06) helped define a realistic limit of quantitation as 0.05 μg/g. Recovery data were quantitative and varied between 81.4 and 98.0% (mean = 89.8%) for each of 6 spiked materials.

An interlaboratory-verified, liquid chromatographic (LC) method is presented for determination of all-racemic α-tocopheryl acetate and retinyl palmitate in infant formula. The extraction procedure uses matrix solid-phase dispersion. A sample is mixed with C18, and the mixture is packed into a reservoir and eluted with selective solvents to extract the analytes. After evaporation and filtration, the sample extract is injected directly into a normal-phase LC system with fluorescence detection. All-racemic α-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing isopropanol at 0.2% (v/v) and 0.125% (v/v), respectively. A nonfortified zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where “x” represents the minimum levels of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in Title 21 of the Code of Federal Regulations, part 107.100. Recoveries of retinyl palmitate ranged from 83.8 to 107%, and those of all-racemic α-tocopheryl acetate ranged from 87.7 to 108%. Two additional laboratories analyzed the ZRM samples at 4 spiking levels with 6 replicates. Recoveries of retinyl palmitate and all-racemic α-tocopheryl acetate ranged from 92.2 to 104% and from 91.7 to 101%, respectively, in the second laboratory. Recoveries of retinyl palmitate and all-racemic α-tocopheryl acetate ranged from 85.3 to 97.0% and from 86.6 to 110%, respectively, in the third laboratory. Relative standard deviations for all 3 laboratories ranged from 0.2 to 7.5% with an average of 2.9%. In addition, each laboratory analyzed a commercial milk- and commercial soy-based infant formula. Excellent agreement in results was obtained between the 3 laboratories for vitamins A and E in all matrixes.

An interlaboratory verified, liquid chromatographic (LC) method is presented for the analysis of all-rac-α-tocopheryl acetate and retinyl palmitate in soy-based infant formula. The extraction procedure uses sample dehydration with magnesium sulfate followed by extraction with isopropanol, hexane–ethyl acetate (85 + 15, v/v). After evaporation and filtration, the sample extract is injected directly onto a normal-phase LC system with fluorescence detection. All-rac-α-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing 0.50% (v/v) and 0.125% (v/v) isopropanol, respectively. A zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where “x” is the minimum level of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in 21 Code of Federal Regulations 107.100. The following recoveries and RSD values represent an average (n = 25) of the 5 levels for each analyte: all-rac-α-tocopheryl acetate, 100% (RSD = 3.5%); retinyl palmitate, 97.2% (RSD = 2.1%). Two additional laboratories analyzed the fortified ZRM samples. Average recoveries (n = 24) of all-rac-α-tocopheryl acetate and retinyl palmitate at 4 levels were all-rac-α-tocopheryl acetate, 99.0% (RSD = 4.0%), and retinyl palmitate, 96.2% (RSD = 1.4%) at the second laboratory. Average recoveries (n = 24) of all-rac-α-tocopheryl acetate and retinyl palmitate at 4 levels were all-rac-α-tocopheryl acetate, 102% (RSD = 1.4%) and retinyl palmitate, 95.7% (RSD = 2.0%) at the third laboratory. In addition, 6 replicates of the same commercial soy-based infant formula powder were run by the 3 laboratories.

A liquid chromatographic method is described for the determination of ergot alkaloids in wheat. Ergonovine, ergotamine, ergocornine, α-ergocryptine, and ergocristine are extracted from wheat with methanol–0.25% concentrated H3PO4 (40 + 60) pH 2.2, cleaned up by using a solid-phase extraction (SPE) disk, and separated by reversed-phase liquid chromatography with fluorescence detection. Ergot alkaloids are basic compounds that form water-soluble salts in acidic aqueous solution. Because ergot alkaloid salts are positively charged, they can be easily and selectively trapped on a negatively charged strong cation-exchange SPE disk. A strong wash solvent, methanol–0.25% concentrated H3PO4 (40 + 60) was used to remove matrix interferences not bonded by ionic interactions with the cation-exchange column. The ergot alkaloids were eluted from the ion-exchange column by adjusting the pH of the elution solvents to slightly basic conditions (pH 9). The SPE disk concentrated and cleanly separated the ergot alkaloids from matrix interferences. Standard calibration curves for ergot alkaloids for the concentration range 0.1–2.0 μg/mL were linear. The SPE disk had a column capacity equivalent to about 1 g extracted wheat. At spiking levels of 2.3–46 ng/g for ergonovine and 20–400 ng/g for ergotamine, ergocornine, α-ergocryptine, and ergocristine, the mean recovery was 88.1% with a coefficient of variation (CV) of 5.33%. The recovery data ranged from 79.1 to 95.9%. Ergonovine had the lowest overall recovery and the largest CV. The method has an estimated reliable limit of detection and limit of quantitation of

A liquid chromatographic (LC) method is described for the determination of vitamin K1 in medical foods. The sample is enzymatically digested with lipase and α-amylase and extracted with 1% sodium bicarbonate solution–isopropanol (1 + 1). After C18 solid-phase extraction, vitamin K1 is separated by nonaqueous reversed-phase LC, converted to the hydroquinone by postcolumn zinc reduction, and quantitated by fluorescence detection. The limit of detection is 8 pg (3 σ), and the limit of quantitation is 27 pg (10 σ) on column. Linear response ranged from 0.1 to 1.0 ng vitamin K1 (r = 0.9999). The mean recovery (n = 38) for all spiking levels was 101.6 ± 2.85%. Analysis of Standard Reference Material 1846, Infant Formula, gave a mean value of 0.95 ± 0.088 mg vitamin K/kg (K or K1?)(n = 31) with a coefficient of variation of 9.26.

A liquid chromatographic method for vitamin K1 in milk-based infant formula is described. The vitamins are extracted from infant formula by matrix solid-phase dispersion and quantitated by reversed-phase chromatography with fluorescence detection. Vitamin K1 is converted to the fluorescent hydroquinone with a postcolumn zinc reductive reactor. The limit of detection is 12 pg, and the limit of quantitation is 38 pg on-column. Linear responses were obtained in the range 0.55-22.1 ng/mL (r2 = 0.9998). Recoveries of vitamin K1 from an analyte-fortified blank material for milk-based infant formula averaged 91.7% (n = 25). The method provides a rapid, specific, and easily controlled assay for vitamin K1 in fortified infant formula.

Fatty acid methyl esters (FAMEs) of pure triglyceride standards, oils, and fat from dry matrixes were formed by transesterification using sodium methoxide in methanol–hexane. FAMEs were produced by direct addition of sodium methoxide¯hexane to samples and heating to simultaneously extract and transesterify acyl lipids. FAMEs were quantitated by capillary gas chromatography (GC) over a fatty acid concentration range of 0 to 1.7 mg/mL (r ≥ 0.9997). Total fat was calculated as the sum of individual fatty acids expressed as triglyceride equivalents, in accordance with nutrition labeling guidelines. Saturated, polyunsaturated, and monounsaturated fats were calculated as sums of individual free fatty acids. Absolute recoveries determined from individual fatty acids in test samples ranged from 69.7 to 106%. Recoveries (relative to the C13:0 internal standard) for individual fatty acids in test samples ranged from 95 to 106%. Reproducibility was constant at each fatty acid level in the reaction mixture (n = 5, coefficient of variation [CV]

A liquid chromatographic method is described for analysis of all-rac-α-tocopheryl acetate and retinyl palmitate in medical food. The vitamins are extracted in isopropyl alcohol and hexane-ethyl acetate without saponification and quantitated by normal-phase chromatography with fluorescence detection. All-rac-α-tocopheryl acetate and retinyl palmitate are chromatographed isocratically with a mobile phase of 0.5% (v/v) and 0.125% (v/v) isopropyl alcohol in hexane, respectively. Recovery studies performed on a medical food zero control reference material (ZRM) fortified with the analytes averaged 99.7% (n = 25) for retinyl palmitate and 101% (n = 25) for all-rac-α-tocopheryl acetate. Coefficients of variation were 0.87-2.63% for retinyl palmitate and 1.42-3.20% for all-rac-α-tocopheryl acetate. The method provides a rapid, specific, and easily controlled assay for analysis of vitamin A and vitamin E in medical foods. Use of chlorinated solvents is avoided.

A liquid chromatographic method is described for analysis of all-rac-α-tocopheryl acetate and retinyl palmitate in medical food. The vitamins are extracted from medical food without saponification by matrix solid-phase dispersion and chromatographed by normal-phase chromatography with fluorescence detection. Retinyl palmitate and all-rac-α-tocopheryl acetate are quantitated isocratically with a mobile phase of 0.125% (v/v) and 0.5% (v/v) isopropyl alcohol in hexane, respectively. Results compared favorably with label declarations on retail medical foods. Recoveries determined on an analyte-fortified zero reference material for a milk-based medical food averaged 98.3% (n = 25) for retinyl palmitate spikes and 95.7% (n = 25) for all-rac-α-tocopheryl acetate spikes. Five concentrations were examined for each analyte, and results were linear (r2 = 0.995 for retinyl palmitate and 0.9998 for all-rac-α-tocopheryl acetate) over the concentration range examined, with coefficients of variation in the range 0.81-4.22%. The method provides a rapid, specific, and easily controlled assay for analysis of retinyl palmitate and all-rac-α-toco-pheryl acetate in fortified medical foods.

A liquid chromatographic method is described for analysis of all-rac-α-tocopheryl acetate, tocopherols, and retinyl palmitate in soy-based infant formula. The vitamins are extracted in isopropyl alcohol and hexane-ethyl acetate without saponification and quantitated by normal-phase chromatography with fluorescence detection. All-rac-α-tocopheryl acetate and retinyl palmitate are quantitated isocratically with mobile phases of 0.5% (v/v) and 0.125% (v/v) isopropyl alcohol in hexane, respectively. Recoveries from zero control reference material soy-based formula averaged 97.2% (n = 25) for retinyl palmitate and 100% (n = 25) for all-rac-α-tocopheryl acetate. Coefficients of variation ranged from 1.21 to 2.86% for retinyl palmitate and from 1.49 to 5.16% for all-rac-α-tocopheryl acetate. The method provides a rapid, specific, and easily controlled assay for analysis of vitamin A and vitamin E in fortified infant formula. Additionally, the method eliminates use of chlorinated solvents.

A zero control reference material (ZRM) for milk and soy-based infant formula was manufactured and characterized. The ZRM was free of retinyl palmitate and all-rac-α-tocopheryl acetate. The composition was similar to commercially available infant formula. The ZRM provides a valuable tool to ascertain method performance.

A liquid chromatographic (LC) method is described for analysis of sucrose polyester (SPE) in salad dressings. SPE is separated from acylglycerols by gel permeation chromatography. The elution time window for SPE was determined with an evaporative light-scattering mass detector. The SPE fraction is collected and quantitated by C18 reversed-phase chromatography with a ternary gradient. The gradient consists initially of 45.5% methylene chloride, 52.5% acetonitrile, and 2.0% isopropyl alcohol. After 5 min, the ratios change to 98.0% methylene chloride and 2.0% isopropyl alcohol and remain unchanged for the next 6 min. The gradient then reverts back to initial conditions and is ready for another injection after a total run time of 17 min. Sucrose octaacetate was used as an internal standard to simplify quantitation. The mean (n = 3) recovery of SPE was 92.4 ± 2.6%. Reversed-phase LC precision was determined by performing 6 replicate analyses of Italian dressing. The mean recovery ± standard deviation and coefficient of variation were 261 ± 9.5 mg SPE/g and 3.6%, respectively.

A reversed-phase ion pair liquid chromatographic method developed for the simultaneous determination of thiamine (B1), riboflavin (B2), and pyridoxine (B6) in perchloric acid extracts of infant formulas was modified to include medical foods. UV detection of B1 and B2 was replaced by fluorescence detection, which resulted in improved sensitivity and specificity. B1 was detected by fluorescence after conversion to thiochrome by a postcolumn reaction with sodium hydroxide and potassium ferricyanide. The method uses a mobile phase of water, acetonitrile, hexanesulfonic acid sodium salt, ammonium hydroxide, and phosphoric acid adjusted to pH 3.6. The column is a 300 × 3.9 mm Nova Pak C18. Limits of detection were 0.05 μg/mL for B1 and B2 and 0.01 μg/mL for B6 by fluorescence detection. The system reproducibility was evaluated by completing 10 repetitive determinations on a medical food that gave a coefficient of variation of 5.9, 6.0, and 10.7% for B1, B2, and B6, respectively. Mean recoveries (n = 10) were 111,96.3, and 113% for B1, B2, and B6, respectively. The results compared favorably with those by AOAC Official Methods 942.23, 940.33, and 961.15 for B1, B2, and B6, respectively.

An ion exchange liquid chromatographic (LC) method using an anion exchange resin column was developed for the determination of niacin in fortified foods. Samples were extracted by autoclaving with H2SO4 (1+1). Florisil open column chromatography was used to remove interferences from the sample extracts. Niacin levels were quantitated by an LC system using a 250 x 4.1 mm Hamilton PRP-X100 column, a mobile phase of 2% glacial acetic acid in water, and UV detection at 254 nm. The limit of detection was 0.11 μg niacin/mL, and the standard curve was linear from 0.24 to 0.80 jag niacin/mL. The system reproducibility was evaluated by completing 10 repetitive analyses on an infant formula and a macaroni product, which gave an average CV of 2.7%. Mean recovery (± standard deviation) was 99.8 ± 7.7 (n = 15). The results compared favorably with those by the AOAC microbiological method.

An Ion pairing reversed-phase liquid chromatographic method developed for multivitamin supplements and premlxes was applied to the simultaneous determination of thiamine, riboflavin, and pyridoxine In perchloric acid extracts of milkand soy-based Infant formulas. The method uses m-hydroxy benzoic acid as Internal standard and a mobile phase consisting of water, acetonltrile, hexanesulfonlc acid sodium salt, and ammonium hydroxide solution, adjusted to pH 3.6 with phosphoric acid. The column Is a 15 cm x 3.9 mm id Nova Pak C18. Limits of detection were 0.15 μg/mL for thiamine and 0.09 μg/mL for riboflavin by UV detection at 254 nm, and 0.010 μg/mL for pyridoxine by fluorescence detection. Mean percent recoveries based on triplicate determinations were 102 ± 1.8,102 ± 3.3, and 101 ± 3.1 for thiamine, riboflavin, and pyridoxine, respectively. The results compared favorably with the AOAC methods for thiamine, riboflavin, and pyridoxine.

A liquid chromatographic method is described for analysis of β-carotene in medical food. The nutrient is extracted from medical food without saponification by matrix solid-phase dispersion and quantitated by isocratic normal-phase chromatography with a Si 60 column and a mobile phase of hexane containing 0.125% (v/v) isopropyl alcohol. The limit of quantitation is 0.02 μg/mL at 436 nm. Standard response was linear over the concentration range of 0.02μ1.0 μg/ml(r2 = 0.99998). Recoveries were determined on a zero control reference material containing added β-carotene at various levels. Recoveries averaged 91.2% (n = 25) with coefficients of variation from 0.50 to 3.10%. The method provides a rapid, specific, sensitive, and easily controlled assay for analysis of β-carotene in fortified medical food. In addition, retinyl palmitate can be assayed simultaneously with an in-line fluorescence detector.

A tri-enzyme digestion procedure using chicken pancreas conjugase, a-amylase, and Pronase® was evaluated to determine Its usefulness In the microbiological quantitation of total folate In foods. Folate values obtained by traditional conjugase digestion were compared to those obtained by the tri-enzyme method for 12 food products that represent diverse matrixes. The tri-enzyme treatment Increased measurable folate from most foods when compared to levels found after conjugase digestion. Largest Increases were noted for tuna fish (51%) and yogurt (33%) after tri-enzyme digestion. For the 12 foods, a mean increase of 19% In measurable folate was obtained with tri-enzyme treatment. The study shows that traditional conjugase treatment does not completely free folate from complex food matrixes before microbiological analysis. Further, as other Investigations have suggested, current accepted methods for folate analysis may be underestimating folate levels In foods

A high pressure liquid chromatographic method was developed using high pressure gel permeation chromatography (HP-GPC) and high pressure reverse phase chromatography (RP-HPLC) for quantitation of retinyl palmitate and β-carotene. HP-GPC was used for fractionation of vitamin A active compounds from oil preliminary to quantitation on nonaqueous RP-HPLC. HP-GPC fractionation was completed on oil and margarine dissolved in methylene chloride by 2 elution passes through 2 μStyragel (100Å) columns connected in series with methylene chloride as the mobile phase. RPHPLC separation of retinyl palmitate and β-carotene was achieved on μBondapak C18 (10 μm), using methylene chloride-acetonitrile ( 3 0 + 7 0 ) . Based on 10 repetitive analyses, recoveries of added β-carotene and retinyl palmitate from vegetable oils were 98.6±2.9 and 95.2±2.6%, respectively. The coefficients of variation were 2.9% for β-carotene and 2.7% for retinyl palmitate. The determination of vitamin A activity in 7 margarine brands with label claims of 10% U.S.RDA/serving revealed that all but one of the margarines contained at least 94% of the label claim. Vitamin A activity in the margarines ranged from 90.6 to 110.8% of the label declaration.

Vitamin A (vitamin A palmitate) and vitamin E (a-tocopheryl acetate) levels were determined in 77 samples of fortified infant formulas manufactured by 4 firms in the United States from 1981 to 1983 and were compared by formulation base (soy, milk) and manufacturing firm. For vitamin A and vitamin E, the mean values (IU/100 kcal) were 454 ± 95 (range 248-614) and 2.0 ± 0.7 (range 1.1-5.0), respectively. No significant differences (a = 0.05) were found in levels (IU/100 kcal) of vitamin A and vitamin E between milk- and soy-based formulas. When the mean vitamin A and vitamin E levels of formulas produced by the various firms were compared on an IU/100 kcal or percent of label declaration basis, significant differences (a = 0.05) were found among firms. Mean vitamin A levels for the various products compared to label declarations ranged from 126% of declared for the ready-to-use formulas to 139% of declared for the powders. Mean vitamin E levels ranged from 97% of declared for ready-to-use formulas to 118% of declared for concentrates. Except for one sample that contained 248 IU vitamin A/100 kcal, the formulas met the requirements of the 1980 Infant Formula Act.